Genes encoding sulfate assimilation proteins

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a sulfate assimilation protein. The invention also relates to the construction of a chimeric gene encoding all or a portion of the sulfate assimilation protein in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the sulfate assimilation protein in a transformed host cell.

This application claims the benefit of U.S. Provisional Application No.60/092,833, filed Jul. 14, 1998.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingsulfate assimilation proteins in plants and seeds.

BACKGROUND OF THE INVENTION

Sulfate assimilation is the process by which environmental sulfur isfixed into organic sulfur for use in cellular metabolism. The two majorend products of this process are the essential amino acids cysteine andmethionine. These amino acids are limiting in food and feed; they cannotbe synthesized by animals and thus must be acquired from plant sources.Increasing the level of these amino acids in feed products is thus ofmajor economic value. Key to that process is increasing the level oforganic sulfur available for cysteine and methionine biosynthesis.

Multiple enzymes are involved in sulfur assimilation. These include:High affinity sulfate transporter and low affinity sulfate transporterproteins which serve to transport sulfur from the outside environmentacross the cell membrane into the cell (Smith et al. (1995) PNAS92(20):9373-9377). Once sulfur is in the cell sulfateadenylyltransferase (ATP sulfurylase) (Bolchia et al. (1999) Plant Mol.Biol. 39(3):527-537) catalyzes the first step in assimilation,converting the inorganic sulfur into an organic form, adenosine-5′phosphosulfate (APS). Next, several enzymes further modify organicsulfur for use in the biosynthesis of cysteine and methionine. Forexample, adenylylsulfate kinase (APS kinase), catalyzes the conversionof APS to the biosynthetic intermediate PAPS (3′-phospho-adenosine-5′phosphosulfate) (Arz et al. (1994) Biochim. Biophy. Acta1218(3):447-452). APS reductase (5′ adenylyl phosphosulphate reductase)is utilized in an alternative pathway, resulting in an inorganic butcellularly bound (bound to a carrier), form of sulfur (sulfite) (Setyaet al. (1996) PNAS 93(23):13383-13388). Sulfite reductase furtherreduces the sulfite, still attached to the carrier, to sulfide andserine O-acetyltransferase converts serine to O-acetylserine, which willserve as the backbone to which the sulfide will be transferred to fromthe carrier to form cysteine (Yonelcura-Sakakibara et al. (1998) J.Biolchem. 124(3):615-621 and Saito et al. (1995) J. Biol. Chem.270(27):16321-16326).

As described, each of these enzymes is involved in sulfate assimilationand the pathway leading to cysteine biosynthesis, which in turn servesas an organic sulfur donor for multiple other pathways in the cell,including methionine biosynthesis. Together or singly these enzymes andthe genes that encode them have utility in overcoming the sulfurlimitations known to exist in crop plants. It may be possible tomodulate the level of sulfur containing compounds in the cell, includingthe nutritionally critical amino acids cysteine and methionine.Specifically, their overexpression using tissue specific promoters willremove the enzyme in question as a possible limiting step, thusincreasing the potential flux through the pathway to the essential aminoacids. This will allow the engineering of plant tissues with increaseslevels of these amino acids, which now often must be added a supplementsto animal feed.

SUMMARY OF THE INVENTION

The instant invention relates to isolated nucleic acid fragmentsencoding sulfate assimilation proteins. Specifically, this inventionconcerns an isolated nucleic acid fragment encoding a sulfite reductaseand an isolated nucleic acid fragment that is substantially similar toan isolated nucleic acid fragment encoding a sulfite reductase. Inaddition, this invention relates to a nucleic acid fragment that iscomplementary to the nucleic acid fragment encoding sulfite reductase.An additional embodiment of the instant invention pertains to apolypeptide encoding all or a substantial portion of a sulfitereductase.

In another embodiment, the instant invention relates to a chimeric geneencoding a sulfite reductase, or to a chimeric gene that comprises anucleic acid fragment that is complementary to a nucleic acid fragmentencoding a sulfite reductase, operably linked to suitable regulatorysequences, wherein expression of the chimeric gene results in productionof levels of the encoded protein in a transformed host cell that isaltered (i.e., increased or decreased) from the level produced in anuntransformed host cell.

In a further embodiment, the instant invention concerns a transformedhost cell comprising in its genome a chimeric gene encoding a sulfitereductase, operably linked to suitable regulatory sequences. Expressionof the chimeric gene results in production of altered levels of theencoded protein in the transformed host cell. The transformed host cellcan be of eukaryotic or prokaryotic origin and include cells derivedfrom higher plants and microorganisms. The invention also includestransformed plants that arise from transformed host cells of higherplants, and seeds derived from such transformed plants.

An additional embodiment of the instant invention concerns a method ofaltering the level of expression of a sulfite reductase in a transformedhost cell comprising: a) transforming a host cell with a chimeric genecomprising a nucleic acid fragment encoding a sulfite reductase; and b)growing the transformed host cell under conditions that are suitable forexpression of the chimeric gene wherein expression of the chimeric generesults in production of altered levels of sulfite reductase in thetransformed host cell.

An addition embodiment of the instant invention concerns a method forobtaining a nucleic acid fragment encoding all or a substantial portionof an amino acid sequence encoding a sulfite reductase.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIGS. 1A-1D show a comparison of the amino acid sequences set forth inSEQ ID NOs:2, 4 and 6 and the Zea mays and Nicotiana tabacum sequences(SEQ ID NOs:7 and 8 respectively).

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 Sulfate Assimilation Proteins SEQ ID NO: Protein CloneDesignation (Nucleotide) (Amino Acid) Sulfite Reductase rlr12.pk0027.d11 2 Sulfite Reductase srm.pk0035.h7 3 4 Sulfite Reductase Contigcomposed of: 5 6 wdk3c.pk006.11 wlm96.pk0012.h1 wlm96.pk028.g9wrl.pk180.b10

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “nucleic acid fragment” is a polymer of RNA or DNAthat is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. A nucleic acid fragment in theform of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA or synthetic DNA.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example. thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-á-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize, under stringent conditions(0.1×SSC, 0.1% SDS, 65° C.), with the nucleic acid fragments disclosedherein.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Preferred are those mucleic acid fragments whose nucleotidesequences encode amino acid sequences that are 95% identical to theamino acid sequences reported herein. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc. Madison, Wis.).Muliple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410). In general, sequenceof ten or more contiguous amino acids or thirty or more contiguousnucleotides is necessary in order to putatively identify a polypeptideor nucleic acid sequence as homologous to a known protein or gene.Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) MolecularBiotechnology 3:225).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expressions”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol 143:277) and particle-acceleratedor “gene gun” transformation technology (Klein et al. (1987) Nature(London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein byreference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of a sulfateassimilation protein have been isolated and identified by comparison ofrandom plant cDNA sequences to public databases containing nucleotideand protein sequences using the BLAST algorithms well known to thoseskilled in the art. The nucleic acid fragments of the instant inventionmay be used to isolate cDNAs and genes encoding homologous proteins fromthe same or other plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction ligase chain reaction).

For example, genes encoding other sulfite reductase enzymes, either ascDNAs or genomic DNAs could be isolated directly by using all or aportion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998) togenerate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673; Loh et al. (1989) Science 243:217). Productsgenerated by the 3′ and 5′ RACE procedures can be combined to generatefull-length cDNAs (Frohman and Martin (1989) Techniques 1:165).

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed polypeptides are presentat higher or lower levels than normal or in cell types or developmentalstages in which they are not normally found. This would have the effectof altering the level of sulfite reductase in those cells. This enzymeis involved in sulfate assimilation and the pathway leading to cysteinebiosynthesis, which in turn serves as an organic sulfur donor formultiple other pathways in the cell. including methionine biosynthesis.This enzyme and the gene(s) that encodes the protein has utility inovercoming the sulfur limitations known to exist in crop plants. It maybe possible to modulate the level of sulfur containing compounds in thecell, including the nutritionally critical amino acids cysteine andmethionine. Specifically, their overexpression using tissue specificpromoters will remove the enzyme in question as a possible limitingstep, thus increasing the potential flux through the pathway to theessential amino acids. This will allow the engineering of plant tissueswith increases levels of these amino acids, which now often must beadded a supplements to animal feed.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of developrment.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can thenconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys.100:1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppresion technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds, and is not an inherentpart of the invention. For example, one can screen by looking forchanges in gene expression by using antibodies specific for the proteinencoded by the gene being suppressed, or one could establish assays thatspecifically measure enzyme activity. A preferred method will be onewhich allows large numbers of samples to be processed rapidly, since itwill be expected that a large number of transformants will be negativefor the desired phenotype.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention iii situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded sulfate assimilation protein. An example of a vector forhigh level expression of the instant polypeptides in a bacterial host isprovided (Example 6).

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4(1):37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Research5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997)Nature Genetics 7:22-28) and Happy Mapping (Dear and Cook (1989) NucleicAcid Res. 17:6795-6807). For these methods, the sequence of a nucleicacid fragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149; Bensen et al. (1995) Plant Cell7:75). The latter approach may be accomplished in two ways. First, shortsegments of the instant nucleic acid fragments may be used in polymerasechain reaction protocols in conjunction with a mutation tag sequenceprimer on DNAs prepared from a population of plants in which Mutatortransposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsiusunless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries: Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various rice, soybean and wheattissues were prepared. The characteristics of the libraries aredescribed below.

TABLE 2 cDNA Libraries from Rice, Soybean and Wheat Library Tissue Clonerlr12 Rice (Oryza sativa L.) leaf (15 days after germination)rlr12.pk0027.d1 12 hours after infection of Magaporthe grisea strain4360-R-62 (AVR2-YAMO); Resistant srm Soybean (Glycine max L.) rootmeristem srm.pk0035.h7 wdk3c Wheat (Triticum aestivum L.) developingkernel, wdk3c.pk006.11 14 days after anthesis wlm96 Wheat (Triticumaestivum L.) seedlings wlm96.pk0012.h1 96 hr after inoculation w/E.graminis wlm96.pk028.g9 wr1 Wheat (Triticum aestivum L.) root; 7 day oldseedling, wr1.pk180.b10 light grown

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP* XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif).The Uni-ZAP* XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids or the insert cDNA sequencesare amplified via polymerase chain reaction using primers specific forvector sequences flanking the inserted cDNA sequences. Amplified insertDNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions togenerate partial cDNA sequences (expressed sequence tags or “ESTs”; seeAdams et al., (1991) Science 252:1651). The resulting ESTs are analyzedusing a Perkin Elmer Model 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

cDNA clones encoding sulfate assimilation proteins were identified byconducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nature Genetics 3:266-272) provided by the NCBI.For convenience, the P-value (probability) of observing a match of acDNA sequence to a sequence contained in the searched databases merelyby chance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Sulfite Reductase

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to sulfitereductase from Zea mays (NCBI Identifier No. gi 2653558) and Nicotianatabacum (NCBI Identifier No. gi 3721540). Shown in Table 3 are the BLASTresults for individual ESTs (“EST”), the sequences of the entire cDNAinserts comprising the indicated cDNA clones (“FIS”), or contigsassembled from two or more ESTs (“Contig”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toZea mays and Nicotiana tabacum Sulfite Reductase Clone Status BLAST pLogScore rlr12.pk0027.d1 FIS >254.00 (gi 2653558) srm.pk0035.h7 FIS >254.00(gi 3721540) Contig composed of: Contig >254.00 (gi 2653558)wdk3c.pk006.11 wlm96.pk0012.h1 wlm96.pk028.g9 wrl.pk180.b10

FIGS. 1A-1D present an alignment of the amino acid sequences set forthin SEQ ID NOs:2, 4 and 6 and the Zea mays and Nicotiana tabacumsequences (SEQ ID NOs:7 and 8 respectively). The data in Table 4represents a calculation of the percent identity of the amino acidsequences set forth in SEQ ID NOs:2, 4 and 6 and the Zea mays andNicotiana tabacum sequences (SEQ ID NOs:7 and 8).

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toZea mays and Nicotiana tabacum Sulfite Reductase SEQ ID NO. PercentIdentity to 2 91% 4 80% 6 89%

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASARGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a sulfite reductase. These sequencesrepresent the first corn, rice, soybean and wheat sequences encodingsulfite reductase.

Example 4 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML 103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcol-SmaI fragment of the plasmid pML 103. Plasmid pML 103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366.The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Pat. Publication 0 242 236) which encodes phosphinothricinacetyl transferase (PAT). The enzyme PAT confers resistance toherbicidal glutamine synthetase inhibitors such as phosphinothricin. Thepat gene in p35S/Ac is under the control of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) andthe 3′ region of the nopaline synthase gene from the T-DNA of the Tiplasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embroys may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, greentransformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430.This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

8 1 2327 DNA Oryza sativa 1 gcacgaggaa gaggaaatat cccactcgct tcgacactctcttccccttc ttctcccgga 60 gaaaacctcg agccaaggcc ccgactccga ctccggtgatgtcggcggcg gtggggggag 120 ccgagttcca cgggttccgt gggggtggcg gcggcgcggcgcagctgcag aggtcgcgga 180 tgctcggaag gccgctccgt gtggcgaccc ctcacgcggcggcccccgct ggcggcggcg 240 ggtcgtcgtc ggccagcata cgcgccgtct ccgcgccactcaagaaggat gcatctgaag 300 ttaagaggag caaggttgag atcatcaagg agaagagcaattttcttcgg taccctttga 360 atgaggaact ggtctcagag gctcccaata ttaatgacagcgctgtccag cttattaaat 420 ttcatggaag ctatcaacag acggaccgtg atgttcgtgggcagaagaat tactcgttta 480 tgctccgaac aaagaatcct tgtggaaaag ttccaaaccagctttacttg gctatggata 540 cgctagctga tgaatttggt attggaacac tccgattgacgaccagacaa acatttcagc 600 tgcatggcgt tcttaagaag aacctgaaga ctgtcatcagcactgttata aagaatatgg 660 gttcatcatt aggtgcctgt ggagatctca acagaaatgtacttgcacct gcagcacctt 720 atgtcaggaa ggatattctt tttgctcaag aaacagcagagaatatcgca gctcttctta 780 caccacaatc tggggcttat tatgacctgt gggtggatggagaaaagata atgtcagccg 840 aagaacctcc tgaggtcacg aaagctcgca atgacaacacatatggaaca aatttccccg 900 attcccctga accaatatat ggcacacagt atctgccaagaaagttcaag attgctgtca 960 ctgtcgctgg agataactct gttgatattc tgaccaatgacatcggtgtt gttgttgttt 1020 cagatagtgc aggagagcct gttggcttca acatttatgttggtggtggc atgggtagga 1080 cacaccgagt ggagaccaca ttccctcgat tggctgatccactgggttat gttcctaagg 1140 aagatatatt gtatgctata aaagcaatag tcgtcacacagagggaaaat gggagaaggg 1200 atgaccgccg atatagcagg atgaagtatc tgattgataactggggaatt gagaagtttc 1260 gggctgaagt cgaaaaatac tatggaaaga agtttgaagattctcgtcct ttgcccgaat 1320 ggcagttcaa cagctaccta ggatggcagg aacagggtgatggaaaatta ttctacggag 1380 tgcatgttga taatggtcgt gtcgcagggc aagcaaagaaaactctacga gagattattg 1440 agaagtacaa tttggaagtt agcattactc caaaccagaatcttatctta tgtgggattg 1500 atcaagcatg gaaagatccc atcacagcag ctcttgctcaatctggcctg ctggaaccaa 1560 aggatgttga tcccctgaat attacttcca tggcatgtcctgccttacca ctgtgccctc 1620 tagcacaaac agaagctgaa cgagggattc tgccgattcttaaacgaatt agagcagttt 1680 ttgataaggt tggcatcaag gaccatgagt cggtagtggtgaggataaca ggctgcccta 1740 atggatgcgc tagaccatat atggcagagg ttggctttgttggtgatggc ccaaacagtt 1800 accagatatg gcttggagga acaccaaacc agagtaccctagctgaaacc tttatgaata 1860 aagtgaagct tcaagatatt gagaaggttt tggaaccattgttttcctat tggaacagca 1920 cacgtcagga aggtgaatct tttggtagct tcacacgccggacgggattt gacaaattga 1980 aagaggtagt gaacaagtgg gcagagtcag catcagctgcatgatggact gctttcgctg 2040 aacaagttga taacaattct gaccacgggt ccaatgcgggcatcgtcaag ggctctcaac 2100 ataactatgt gagcattgca ggagaaaatt ttgtcaatttcgttgacaag attgaggact 2160 cgccgactcg ggtttggaat cgttcgttca gataattaatcaaaattttt cgtgtactct 2220 ggtttgagaa aaaaaaatgt gcttatgaga aaacaaaaggaaccctggct gtttactttg 2280 gaataaattg cttggaaagt gtactgaata aaaaaaaaaaaaaaaaa 2327 2 673 PRT Oryza sativa 2 Thr Arg Lys Arg Lys Tyr Pro ThrArg Phe Asp Thr Leu Phe Pro Phe 1 5 10 15 Phe Ser Arg Arg Lys Pro ArgAla Lys Ala Pro Thr Pro Thr Pro Val 20 25 30 Met Ser Ala Ala Val Gly GlyAla Glu Phe His Gly Phe Arg Gly Gly 35 40 45 Gly Gly Gly Ala Ala Gln LeuGln Arg Ser Arg Met Leu Gly Arg Pro 50 55 60 Leu Arg Val Ala Thr Pro HisAla Ala Ala Pro Ala Gly Gly Gly Gly 65 70 75 80 Ser Ser Ser Ala Ser IleArg Ala Val Ser Ala Pro Leu Lys Lys Asp 85 90 95 Ala Ser Glu Val Lys ArgSer Lys Val Glu Ile Ile Lys Glu Lys Ser 100 105 110 Asn Phe Leu Arg TyrPro Leu Asn Glu Glu Leu Val Ser Glu Ala Pro 115 120 125 Asn Ile Asn AspSer Ala Val Gln Leu Ile Lys Phe His Gly Ser Tyr 130 135 140 Gln Gln ThrAsp Arg Asp Val Arg Gly Gln Lys Asn Tyr Ser Phe Met 145 150 155 160 LeuArg Thr Lys Asn Pro Cys Gly Lys Val Pro Asn Gln Leu Tyr Leu 165 170 175Ala Met Asp Thr Leu Ala Asp Glu Phe Gly Ile Gly Thr Leu Arg Leu 180 185190 Thr Thr Arg Gln Thr Phe Gln Leu His Gly Val Leu Lys Lys Asn Leu 195200 205 Lys Thr Val Ile Ser Thr Val Ile Lys Asn Met Gly Ser Ser Leu Gly210 215 220 Ala Cys Gly Asp Leu Asn Arg Asn Val Leu Ala Pro Ala Ala ProTyr 225 230 235 240 Val Arg Lys Asp Ile Leu Phe Ala Gln Glu Thr Ala GluAsn Ile Ala 245 250 255 Ala Leu Leu Thr Pro Gln Ser Gly Ala Tyr Tyr AspLeu Trp Val Asp 260 265 270 Gly Glu Lys Ile Met Ser Ala Glu Glu Pro ProGlu Val Thr Lys Ala 275 280 285 Arg Asn Asp Asn Thr Tyr Gly Thr Asn PhePro Asp Ser Pro Glu Pro 290 295 300 Ile Tyr Gly Thr Gln Tyr Leu Pro ArgLys Phe Lys Ile Ala Val Thr 305 310 315 320 Val Ala Gly Asp Asn Ser ValAsp Ile Leu Thr Asn Asp Ile Gly Val 325 330 335 Val Val Val Ser Asp SerAla Gly Glu Pro Val Gly Phe Asn Ile Tyr 340 345 350 Val Gly Gly Gly MetGly Arg Thr His Arg Val Glu Thr Thr Phe Pro 355 360 365 Arg Leu Ala AspPro Leu Gly Tyr Val Pro Lys Glu Asp Ile Leu Tyr 370 375 380 Ala Ile LysAla Ile Val Val Thr Gln Arg Glu Asn Gly Arg Arg Asp 385 390 395 400 AspArg Arg Tyr Ser Arg Met Lys Tyr Leu Ile Asp Asn Trp Gly Ile 405 410 415Glu Lys Phe Arg Ala Glu Val Glu Lys Tyr Tyr Gly Lys Lys Phe Glu 420 425430 Asp Ser Arg Pro Leu Pro Glu Trp Gln Phe Asn Ser Tyr Leu Gly Trp 435440 445 Gln Glu Gln Gly Asp Gly Lys Leu Phe Tyr Gly Val His Val Asp Asn450 455 460 Gly Arg Val Ala Gly Gln Ala Lys Lys Thr Leu Arg Glu Ile IleGlu 465 470 475 480 Lys Tyr Asn Leu Glu Val Ser Ile Thr Pro Asn Gln AsnLeu Ile Leu 485 490 495 Cys Gly Ile Asp Gln Ala Trp Lys Asp Pro Ile ThrAla Ala Leu Ala 500 505 510 Gln Ser Gly Leu Leu Glu Pro Lys Asp Val AspPro Leu Asn Ile Thr 515 520 525 Ser Met Ala Cys Pro Ala Leu Pro Leu CysPro Leu Ala Gln Thr Glu 530 535 540 Ala Glu Arg Gly Ile Leu Pro Ile LeuLys Arg Ile Arg Ala Val Phe 545 550 555 560 Asp Lys Val Gly Ile Lys AspHis Glu Ser Val Val Val Arg Ile Thr 565 570 575 Gly Cys Pro Asn Gly CysAla Arg Pro Tyr Met Ala Glu Val Gly Phe 580 585 590 Val Gly Asp Gly ProAsn Ser Tyr Gln Ile Trp Leu Gly Gly Thr Pro 595 600 605 Asn Gln Ser ThrLeu Ala Glu Thr Phe Met Asn Lys Val Lys Leu Gln 610 615 620 Asp Ile GluLys Val Leu Glu Pro Leu Phe Ser Tyr Trp Asn Ser Thr 625 630 635 640 ArgGln Glu Gly Glu Ser Phe Gly Ser Phe Thr Arg Arg Thr Gly Phe 645 650 655Asp Lys Leu Lys Glu Val Val Asn Lys Trp Ala Glu Ser Ala Ser Ala 660 665670 Ala 3 2408 DNA Glycine max 3 tgatgacgac gtcttttgga ccagccaccacctcagcgcc gcttaaggac cacaaagttc 60 agatcccaag cttccatggc ttgaggtcttcctccgcctc tgctctcccc cgcaatgccc 120 tctcccttcc ttcatccact cgctctctctccctcatacg tgctgtttcc acgcctgcgc 180 agtctgaaac tgccactgtc aagcgcagcaaagtcgaaat attcaaagaa caaagcaatt 240 tcataagata tcctcttaac gaggacattttgacggatgc tcctaatata agtgaagccg 300 ccactcaatt gatcaagttt catggtagctatcaacagta caatagagag gagcgtggtt 360 ccagaagcta ctctttcatg atacgcactaagaatccatg cgggaaggtt tccaaccaac 420 tttacctcac catggatgat cttgctgaccagtttgggat tgggacgctt cgcttgacca 480 ccaggcagac gtttcagctc catggtgttctcaagaagga ccttaaaaca gtcatgggta 540 ccattattag gaacatgggc tcgactcttggtgcttgtgg cgacctaaac aggaatgtgc 600 ttgctcctgc agctcccctt gcaagaaaagattacctctt tgctcaacaa actgctgaga 660 acattgctgc gctcctcgct cctcagtctggtttctacta tgatatttgg gtggatgggg 720 aaaagatttt gacatcagaa ccacctgaagtagttcaggc acgaaatgac aattctcatg 780 gtacaaactt cccggattcc cccgagcccatctatggaac tcagttcttg ccaaggaaat 840 tcaaaattgc tgttactgtg ccaactgataactccgtgga cattctcaca aatgatattg 900 gtgttgttgt tgttaccgat gacgatggggagcctcaagg gttcaacata tatgttggtg 960 gtggaatggg aagaactcat aggttggaaaccacttttcc tcgcttggca gaaccaatag 1020 gttacgtacc aaaggaggat attttgtatgcagtgaaagc aattgttgtt acacaacgag 1080 aaaatgggag aagagatgac cgcaagtatagtagattgaa atatttgata agctcttggg 1140 gaattgaaaa gtttagaagt gtagttgagcaatattatgg caagaaattt gaacctttcc 1200 gtgcattgcc agaatgggaa tttaaaagttatcttgggtg gcatgaacag ggcgatggca 1260 aactttttta tggtcttcat gttgataatggtcgtattgg tggaaacatg aaaaagacat 1320 tgagggaggt tatcgagaag tataatttgaatgtaagaat cactccaaat cagaatatca 1380 tcttgactga tgttcgtgct gcatggaagcgtcccattac aaccacgctt gctcaagctg 1440 gtttgctgca acctagattt gtagatcccctcaacataac agcaatggca tgccctgctt 1500 tcccattatg tcctctggca attactgaagctgaacgtgg gatacctaac atacttaagc 1560 ggattcgtga tgtttttgat aaggttggcctgaagtatag tgagtctgtg gttgtaagga 1620 taactggctg ccctaatggt tgtgctagaccatacatggc tgaacttgga ctagttggtg 1680 atggtccaaa tagctatcag atttggcttggaggaaacca taaacaaaca tcattagctc 1740 gaagtttcat ggacagggtg aagattctagaccttgaaaa agttttggag cctttgtttt 1800 attattggaa gcaaaagcgt caatctaaagaatcatttgg tgacttcaca aaccgaatgg 1860 gatttgagaa gcttaaagaa tatattgagaaatgggaggg tccagtggta gcaccatcac 1920 gccacaacct caagcttttt gctgacaaggagacatatga atcaatggat gcattagcaa 1980 agcttcaaaa caagactgct catcagttggccatggaagt tatccgtaat tatgttgctt 2040 ccaaccaaaa tggaaaaggc gaatgatttcatttttactt aacgaaggaa gatgtatgtg 2100 atgttgcttt atggttgaca ggaatggtggataggcaact gaacacaact ctgttgttac 2160 tgtgtggtaa ctcgggttcg actaaactaatgtttgggtt tttgtttttt tatctgaaac 2220 ggctttccgt agaatctttt ggttcatcatttagatcgag tttctgaaca taaaataagc 2280 ctttctgtca tttctgtatc caattttgggtgttcgacag cttggttttt cttcacaata 2340 atcttgctag ccaagacctt tttccgattttgcgcttgct ccaataaagt tcattaatca 2400 ggatttgt 2408 4 687 PRT Glycinemax 4 Met Thr Thr Ser Phe Gly Pro Ala Thr Thr Ser Ala Pro Leu Lys Asp 15 10 15 His Lys Val Gln Ile Pro Ser Phe His Gly Leu Arg Ser Ser Ser Ala20 25 30 Ser Ala Leu Pro Arg Asn Ala Leu Ser Leu Pro Ser Ser Thr Arg Ser35 40 45 Leu Ser Leu Ile Arg Ala Val Ser Thr Pro Ala Gln Ser Glu Thr Ala50 55 60 Thr Val Lys Arg Ser Lys Val Glu Ile Phe Lys Glu Gln Ser Asn Phe65 70 75 80 Ile Arg Tyr Pro Leu Asn Glu Asp Ile Leu Thr Asp Ala Pro AsnIle 85 90 95 Ser Glu Ala Ala Thr Gln Leu Ile Lys Phe His Gly Ser Tyr GlnGln 100 105 110 Tyr Asn Arg Glu Glu Arg Gly Ser Arg Ser Tyr Ser Phe MetIle Arg 115 120 125 Thr Lys Asn Pro Cys Gly Lys Val Ser Asn Gln Leu TyrLeu Thr Met 130 135 140 Asp Asp Leu Ala Asp Gln Phe Gly Ile Gly Thr LeuArg Leu Thr Thr 145 150 155 160 Arg Gln Thr Phe Gln Leu His Gly Val LeuLys Lys Asp Leu Lys Thr 165 170 175 Val Met Gly Thr Ile Ile Arg Asn MetGly Ser Thr Leu Gly Ala Cys 180 185 190 Gly Asp Leu Asn Arg Asn Val LeuAla Pro Ala Ala Pro Leu Ala Arg 195 200 205 Lys Asp Tyr Leu Phe Ala GlnGln Thr Ala Glu Asn Ile Ala Ala Leu 210 215 220 Leu Ala Pro Gln Ser GlyPhe Tyr Tyr Asp Ile Trp Val Asp Gly Glu 225 230 235 240 Lys Ile Leu ThrSer Glu Pro Pro Glu Val Val Gln Ala Arg Asn Asp 245 250 255 Asn Ser HisGly Thr Asn Phe Pro Asp Ser Pro Glu Pro Ile Tyr Gly 260 265 270 Thr GlnPhe Leu Pro Arg Lys Phe Lys Ile Ala Val Thr Val Pro Thr 275 280 285 AspAsn Ser Val Asp Ile Leu Thr Asn Asp Ile Gly Val Val Val Val 290 295 300Thr Asp Asp Asp Gly Glu Pro Gln Gly Phe Asn Ile Tyr Val Gly Gly 305 310315 320 Gly Met Gly Arg Thr His Arg Leu Glu Thr Thr Phe Pro Arg Leu Ala325 330 335 Glu Pro Ile Gly Tyr Val Pro Lys Glu Asp Ile Leu Tyr Ala ValLys 340 345 350 Ala Ile Val Val Thr Gln Arg Glu Asn Gly Arg Arg Asp AspArg Lys 355 360 365 Tyr Ser Arg Leu Lys Tyr Leu Ile Ser Ser Trp Gly IleGlu Lys Phe 370 375 380 Arg Ser Val Val Glu Gln Tyr Tyr Gly Lys Lys PheGlu Pro Phe Arg 385 390 395 400 Ala Leu Pro Glu Trp Glu Phe Lys Ser TyrLeu Gly Trp His Glu Gln 405 410 415 Gly Asp Gly Lys Leu Phe Tyr Gly LeuHis Val Asp Asn Gly Arg Ile 420 425 430 Gly Gly Asn Met Lys Lys Thr LeuArg Glu Val Ile Glu Lys Tyr Asn 435 440 445 Leu Asn Val Arg Ile Thr ProAsn Gln Asn Ile Ile Leu Thr Asp Val 450 455 460 Arg Ala Ala Trp Lys ArgPro Ile Thr Thr Thr Leu Ala Gln Ala Gly 465 470 475 480 Leu Leu Gln ProArg Phe Val Asp Pro Leu Asn Ile Thr Ala Met Ala 485 490 495 Cys Pro AlaPhe Pro Leu Cys Pro Leu Ala Ile Thr Glu Ala Glu Arg 500 505 510 Gly IlePro Asn Ile Leu Lys Arg Ile Arg Asp Val Phe Asp Lys Val 515 520 525 GlyLeu Lys Tyr Ser Glu Ser Val Val Val Arg Ile Thr Gly Cys Pro 530 535 540Asn Gly Cys Ala Arg Pro Tyr Met Ala Glu Leu Gly Leu Val Gly Asp 545 550555 560 Gly Pro Asn Ser Tyr Gln Ile Trp Leu Gly Gly Asn His Lys Gln Thr565 570 575 Ser Leu Ala Arg Ser Phe Met Asp Arg Val Lys Ile Leu Asp LeuGlu 580 585 590 Lys Val Leu Glu Pro Leu Phe Tyr Tyr Trp Lys Gln Lys ArgGln Ser 595 600 605 Lys Glu Ser Phe Gly Asp Phe Thr Asn Arg Met Gly PheGlu Lys Leu 610 615 620 Lys Glu Tyr Ile Glu Lys Trp Glu Gly Pro Val ValAla Pro Ser Arg 625 630 635 640 His Asn Leu Lys Leu Phe Ala Asp Lys GluThr Tyr Glu Ser Met Asp 645 650 655 Ala Leu Ala Lys Leu Gln Asn Lys ThrAla His Gln Leu Ala Met Glu 660 665 670 Val Ile Arg Asn Tyr Val Ala SerAsn Gln Asn Gly Lys Gly Glu 675 680 685 5 1152 DNA Triticum aestivum 5ggcgatagtt gttacacaga gggaaaatgg aagaagagat gaccgcaggt atagcagact 60gaagtatctg cttgacagct ggggaattga caagtttcgg gccgaagctg aaaaatacta 120tgggaagaag tttgaagatt tccgcccatt gccggaatgg cagttcaaca gctaccttgg 180gtggcaggag cagggtgatg gtaaattatt ctatggagtg catgttgata atggtcgtct 240tggggggcaa gcaaagaaaa ctctgcgaga gataattgag aagtatagct tggatgttag 300tattactcca aaccaaaacc ttatcttatg tggggttgat caggcatgga gagaacccat 360aactgcagct cttgctcaag ctggcctgtt ggaaccaaag gatgttgatc tcctgaacat 420aacctccatg gcatgccctg ccttacctct gtgccctcta gcacaaacag aagctgaacg 480agggatcctg ccaattctta aacgaattag agcagttttt gacaaggttg gtatcaagga 540tgaggagtct gtagtggtga ggataactgg ctgccccaat ggatgcgcca gaccatatat 600ggcagaggtt ggctttgttg gtgacggccc aaacagctac cagatatggc ttggaggaac 660accaaaccag accaccttgg cagagacgtt tatgaataaa gtgaagcttc aagatattga 720gaaagttttg gaaccactgt tttcctattg gaatagcacg cgccaggaag gcgaatcctt 780tggaagcttc acaaaccgaa tgggatttga gcaactgaag gaggtggtga acaagtggga 840ggggtcagcg tcagccgcat gagagttgtc tttgctggac aaatcccagc accatttttg 900ctggggtgag aatctggtgg cgaccaatta ctccacggat tacttttata taaaaactta 960ggagaggagg aggaaacctg tcaattccgt tgacaggcgg aggacgaaga aagagccggg 1020tctgaagaag ttgctccttt gtgtttgttg tgaggtttta tttttttgtg tgtacttgta 1080tggataactc cgttggcccc tttgtttagc ctgagaataa attccttgca aaaaaaaaaa 1140aaaaaaaaaa aa 1152 6 286 PRT Triticum aestivum 6 Ala Ile Val Val Thr GlnArg Glu Asn Gly Arg Arg Asp Asp Arg Arg 1 5 10 15 Tyr Ser Arg Leu LysTyr Leu Leu Asp Ser Trp Gly Ile Asp Lys Phe 20 25 30 Arg Ala Glu Ala GluLys Tyr Tyr Gly Lys Lys Phe Glu Asp Phe Arg 35 40 45 Pro Leu Pro Glu TrpGln Phe Asn Ser Tyr Leu Gly Trp Gln Glu Gln 50 55 60 Gly Asp Gly Lys LeuPhe Tyr Gly Val His Val Asp Asn Gly Arg Leu 65 70 75 80 Gly Gly Gln AlaLys Lys Thr Leu Arg Glu Ile Ile Glu Lys Tyr Ser 85 90 95 Leu Asp Val SerIle Thr Pro Asn Gln Asn Leu Ile Leu Cys Gly Val 100 105 110 Asp Gln AlaTrp Arg Glu Pro Ile Thr Ala Ala Leu Ala Gln Ala Gly 115 120 125 Leu LeuGlu Pro Lys Asp Val Asp Leu Leu Asn Ile Thr Ser Met Ala 130 135 140 CysPro Ala Leu Pro Leu Cys Pro Leu Ala Gln Thr Glu Ala Glu Arg 145 150 155160 Gly Ile Leu Pro Ile Leu Lys Arg Ile Arg Ala Val Phe Asp Lys Val 165170 175 Gly Ile Lys Asp Glu Glu Ser Val Val Val Arg Ile Thr Gly Cys Pro180 185 190 Asn Gly Cys Ala Arg Pro Tyr Met Ala Glu Val Gly Phe Val GlyAsp 195 200 205 Gly Pro Asn Ser Tyr Gln Ile Trp Leu Gly Gly Thr Pro AsnGln Thr 210 215 220 Thr Leu Ala Glu Thr Phe Met Asn Lys Val Lys Leu GlnAsp Ile Glu 225 230 235 240 Lys Val Leu Glu Pro Leu Phe Ser Tyr Trp AsnSer Thr Arg Gln Glu 245 250 255 Gly Glu Ser Phe Gly Ser Phe Thr Asn ArgMet Gly Phe Glu Gln Leu 260 265 270 Lys Glu Val Val Asn Lys Trp Glu GlySer Ala Ser Ala Ala 275 280 285 7 635 PRT Zea mays 7 Met Ser Gly Ala IleGly Gly Ala Glu Val His Gly Phe Arg Gly Ala 1 5 10 15 Ala Ala Gln LeuPro Arg Ser Arg Val Leu Gly Arg Pro Ile Arg Val 20 25 30 Ala Pro Pro AlaAla Ala Arg Pro Gly Gly Ala Ser Ala Gly Ser Ile 35 40 45 Arg Ala Val SerAla Pro Ala Lys Lys Asp Ala Ser Glu Val Lys Arg 50 55 60 Ser Lys Val GluIle Ile Lys Glu Lys Ser Asn Phe Leu Arg Tyr Pro 65 70 75 80 Leu Asn GluGlu Leu Val Ser Glu Ala Pro Asn Ile Asn Glu Ser Ala 85 90 95 Val Gln LeuIle Lys Phe His Gly Ser Tyr Gln Gln Thr Asp Arg Asp 100 105 110 Val ArgGly Gln Lys Asn Tyr Ser Phe Met Leu Arg Thr Lys Asn Pro 115 120 125 CysGly Lys Val Pro Asn Gln Leu Tyr Leu Ala Met Asp Thr Leu Ala 130 135 140Asp Glu Phe Gly Ile Gly Thr Leu Arg Leu Thr Thr Arg Gln Thr Phe 145 150155 160 Gln Leu His Gly Val Leu Lys Lys Asn Leu Lys Thr Val Leu Ser Thr165 170 175 Val Ile Lys Asn Met Gly Ser Thr Leu Gly Ala Cys Gly Asp LeuAsn 180 185 190 Arg Asn Val Leu Ala Pro Ala Ala Pro Tyr Val Lys Lys AspIle Leu 195 200 205 Phe Ala Gln Gln Thr Ala Glu Asn Ile Ala Ala Leu LeuThr Pro Gln 210 215 220 Ser Gly Ala Tyr Tyr Asp Leu Trp Val Asp Gly GluLys Ile Met Ser 225 230 235 240 Ala Glu Glu Pro Pro Glu Val Thr Lys AlaArg Asn Asp Asn Ser His 245 250 255 Gly Thr Asn Phe Pro Asp Ser Pro GluPro Ile Tyr Gly Thr Gln Tyr 260 265 270 Leu Pro Arg Lys Phe Lys Val AlaVal Thr Ala Ala Gly Asp Asn Ser 275 280 285 Val Asp Ile Leu Thr Asn AspIle Gly Val Val Val Val Ser Asp Asp 290 295 300 Ala Gly Glu Pro Ile GlyPhe Asn Ile Tyr Val Gly Gly Gly Met Gly 305 310 315 320 Arg Thr His ArgVal Glu Thr Thr Phe Pro Arg Leu Ala Asp Pro Leu 325 330 335 Gly Tyr ValPro Lys Glu Asp Ile Leu Tyr Ala Ile Lys Ala Ile Val 340 345 350 Val ThrGln Arg Glu Asn Gly Arg Arg Asp Asp Arg Lys Tyr Ser Arg 355 360 365 MetLys Tyr Met Ile Asp Arg Trp Gly Ile Asp Arg Phe Arg Ala Glu 370 375 380Val Glu Lys Tyr Tyr Gly Lys Lys Phe Glu Ser Phe Arg Pro Leu Pro 385 390395 400 Glu Trp Gln Phe Asn Ser Tyr Leu Gly Trp Gln Glu Gln Gly Asp Gly405 410 415 Lys Leu Phe Tyr Gly Val His Val Asp Asn Gly Arg Val Gly GlyGln 420 425 430 Ala Lys Lys Thr Leu Arg Glu Ile Ile Glu Lys Tyr Asn LeuAsp Val 435 440 445 Ser Ile Thr Pro Asn Gln Asn Leu Ile Leu Cys Gly IleAsp Gln Ala 450 455 460 Trp Arg Glu Pro Ile Thr Thr Ala Leu Ala Gln AlaGly Leu Leu Glu 465 470 475 480 Pro Lys Asp Val Asp Pro Leu Asn Leu ThrAla Met Ala Cys Pro Ala 485 490 495 Leu Pro Leu Cys Pro Leu Ala Gln ThrGlu Ala Glu Arg Gly Ile Leu 500 505 510 Pro Ile Leu Lys Arg Ile Arg AlaVal Phe Asn Lys Val Gly Ile Lys 515 520 525 Asp Ser Glu Ser Val Val ValArg Ile Thr Gly Cys Pro Asn Gly Cys 530 535 540 Ala Arg Pro Tyr Met AlaGlu Leu Gly Phe Val Gly Asp Gly Pro Lys 545 550 555 560 Ser Tyr Gln IleTrp Leu Gly Gly Thr Pro Asn Gln Ser Thr Leu Ala 565 570 575 Glu Ser PheMet Asp Lys Val Lys Leu Asp Asp Ile Glu Lys Val Leu 580 585 590 Glu ProLeu Phe Thr Tyr Trp Asn Gly Thr Arg Gln Glu Gly Glu Ser 595 600 605 PheGly Ser Phe Thr Asn Arg Thr Gly Phe Asp Lys Leu Lys Glu Val 610 615 620Val Asn Lys Trp Ala Glu Ser Pro Ser Ala Ala 625 630 635 8 693 PRTNicotiana tabacum 8 Met Thr Thr Ser Phe Gly Ala Ala Ile Asn Ile Ala ValAla Asp Asp 1 5 10 15 Pro Asn Pro Lys Leu Gln Ile His Asn Phe Ser GlyLeu Lys Ser Thr 20 25 30 Ser Asn Ser Leu Leu Leu Ser Arg Arg Leu His ValPhe Gln Ser Phe 35 40 45 Ser Pro Ser Asn Pro Ser Ser Ile Val Arg Ala ValSer Thr Pro Ala 50 55 60 Lys Pro Ala Ala Val Glu Pro Lys Arg Ser Lys ValGlu Ile Phe Lys 65 70 75 80 Glu Gln Ser Asn Phe Ile Arg Tyr Pro Leu AsnGlu Glu Ile Leu Asn 85 90 95 Asp Ala Pro Asn Ile Asn Glu Ala Ala Thr GlnLeu Ile Lys Phe His 100 105 110 Gly Ser Tyr Met Gln Tyr Asp Arg Asp GluArg Gly Gly Arg Ser Tyr 115 120 125 Ser Phe Met Leu Arg Thr Lys Asn ProGly Gly Glu Val Pro Asn Arg 130 135 140 Leu Tyr Leu Val Met Asp Asp LeuAla Asp Gln Phe Gly Ile Gly Thr 145 150 155 160 Leu Arg Leu Thr Thr ArgGln Thr Phe Gln Leu His Gly Val Leu Lys 165 170 175 Lys Asn Leu Lys ThrVal Met Ser Thr Ile Ile Lys Asn Met Gly Ser 180 185 190 Thr Leu Gly AlaCys Gly Asp Leu Asn Arg Asn Val Leu Ala Pro Ala 195 200 205 Ala Pro PheAla Lys Lys Asp Tyr Met Phe Ala Lys Gln Thr Ala Asp 210 215 220 Asn IleAla Ala Leu Leu Thr Pro Gln Ser Gly Phe Tyr Tyr Asp Val 225 230 235 240Trp Val Asp Gly Glu Lys Val Met Thr Ala Glu Pro Pro Glu Val Val 245 250255 Lys Ala Arg Asn Asp Asn Ser His Gly Thr Asn Phe Pro Asp Ser Pro 260265 270 Glu Pro Ile Tyr Gly Thr Gln Phe Leu Pro Arg Lys Phe Lys Ile Ala275 280 285 Val Thr Val Pro Thr Asp Asn Ser Val Asp Ile Phe Thr Asn AspIle 290 295 300 Gly Val Val Val Val Ser Asn Glu Asp Gly Glu Pro Gln GlyPhe Asn 305 310 315 320 Ile Tyr Val Gly Gly Gly Met Gly Arg Thr His ArgMet Glu Thr Thr 325 330 335 Phe Pro Arg Leu Ala Glu Pro Leu Gly Tyr ValPro Lys Glu Asp Ile 340 345 350 Leu Tyr Ala Val Lys Ala Ile Val Val ThrGln Arg Glu Asn Gly Arg 355 360 365 Arg Asp Asp Arg Arg Tyr Ser Arg LeuLys Tyr Leu Leu Ser Ser Trp 370 375 380 Gly Ile Glu Lys Phe Arg Ser ValThr Glu Gln Tyr Tyr Gly Lys Lys 385 390 395 400 Phe Gln Pro Cys Arg GluLeu Pro Glu Trp Glu Phe Lys Ser Tyr Leu 405 410 415 Gly Trp His Glu AlaGly Asp Gly Ser Leu Phe Cys Gly Leu His Val 420 425 430 Asp Asn Gly ArgVal Lys Gly Ala Met Lys Lys Ala Leu Arg Glu Val 435 440 445 Ile Glu LysTyr Asn Leu Asn Val Arg Leu Thr Pro Asn Gln Asn Ile 450 455 460 Ile LeuCys Asn Ile Arg Gln Ala Trp Lys Arg Pro Ile Thr Thr Val 465 470 475 480Leu Ala Gln Gly Gly Leu Leu Gln Pro Arg Tyr Val Asp Pro Leu Asn 485 490495 Leu Thr Ala Met Ala Cys Pro Ala Phe Pro Leu Cys Pro Leu Ala Ile 500505 510 Thr Glu Ala Glu Arg Gly Ile Pro Asp Ile Leu Lys Arg Val Arg Ala515 520 525 Ile Phe Glu Arg Val Gly Leu Lys Tyr Ser Glu Ser Val Val IleArg 530 535 540 Ile Thr Gly Cys Pro Asn Gly Cys Ala Arg Pro Tyr Met AlaGlu Leu 545 550 555 560 Gly Leu Val Gly Asp Gly Pro Asn Ser Tyr Gln IleTrp Leu Gly Gly 565 570 575 Thr Pro Asn Gln Thr Ser Leu Ala Lys Thr PheLys Asp Lys Leu Lys 580 585 590 Val Gln Asp Leu Glu Lys Val Leu Glu ProLeu Phe Phe His Trp Arg 595 600 605 Arg Lys Arg Gln Ser Lys Glu Ser PheGly Asp Phe Thr Asn Arg Met 610 615 620 Gly Phe Glu Lys Leu Gly Glu PheVal Glu Lys Trp Glu Gly Ile Pro 625 630 635 640 Glu Ser Ser Ser Arg TyrAsn Leu Lys Leu Phe Ala Asp Arg Glu Thr 645 650 655 Tyr Glu Ala Met AspAla Leu Ala Ser Ile Gln Asp Lys Asn Ala His 660 665 670 Gln Leu Ala IleGlu Val Val Arg Asn Tyr Val Ala Ser Gln Gln Asn 675 680 685 Gly Lys SerMet Asp 690

What is claimed is:
 1. An isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide having sulfite reductaseactivity, wherein the polypeptide has an amino acid sequence of at least95% sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:4, or (b) a complement of the nucleotide sequence,wherein the complement and the nucleotide sequence consist of the samenumber of nucleotides and are 100% complementary.
 2. The polynucleotideof claim 1, wherein the amino acid sequence of the polypeptide comprisesSEQ ID NO:4.
 3. The polynucleotide of claim 1 wherein the nucleotidesequence comprises SEQ ID NO:3.
 4. A vector comprising thepolynucleotide of claim
 1. 5. A recombinant DNA construct comprising thepolynucleotide of claim 1 operably linked to at least one regulatorysequence.
 6. A method for transforming a cell, comprising transforming acell with the polynucleotide of claim
 1. 7. A cell comprising therecombinant DNA construct of claim
 5. 8. A method for producing a plantcomprising transforming a plant cell with the polynucleotide of claim 1and regenerating a plant from the transformed plant cell.
 9. A plantcomprising the recombinant DNA construct of claim
 5. 10. A seedcomprising the recombinant DNA construct of claim
 5. 11. A method forproduction of a polypeptide having sulfite reductase activity comprisingthe steps of cultivating the cell of claim 7 under conditions that allowfor the synthesis of the polypeptide and isolating the polypeptide fromthe cultivated cells, from culture medium, or from both the cultivatedcells and the culture medium.